title: High-throughput functional assays of IP3-evoked Ca2+ release.
abstract: This protocol describes procedures for high-throughput functional analyses of inositol 1,4,5-trisphosphate receptors (IP3Rs) in permeabilized cells. The methods are applicable to native IP3Rs in a variety of cells and to recombinant IP3Rs stably expressed in DT40 cells in which gene disruption has abolished expression of endogenous IP3Rs. A low-affinity Ca(2+)-indicator (Mag-Fluo-4) trapped within the endoplasmic reticulum (ER) of permeabilized cells is used to report changes in luminal free Ca(2+) concentration. A fluorescence plate-reader equipped to allow automated additions permits rapid measurements of the Ca(2+) release evoked by IP3R. The procedure can be completed in 2-3 h.
authors.affiliation: Department of Pharmacology, University of Cambridge, Cambridge CB2 1PD, United Kingdom.
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Tovey
authors.forename: Colin W
authors.initials: CW
authors.lastname: Taylor
mesh_terms: D002118:Calcium / Q000378:metabolism*; D002413:Cations, Divalent / Q000378:metabolism; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000187:drug effects* / Q000378:metabolism*
chemical_list: D002413:Cations, Divalent; D053496:Inositol 1,4,5-Trisphosphate Receptors; D015544:Inositol 1,4,5-Trisphosphate; D002118:Calcium